A critical driving force behind research in bioprocess science and engineering continues to be the demand for fast and accurate analytical information that can be used, for example, to evaluate the interactions between biological systems and bioprocess operations. One significant challenge is to carry out large numbers of experiments rapidly and effectively. This issue is of particular importance since many of the advances in molecular biology now lead to large numbers of potential biological systems that contain evolved biocatalysts, new pathway designs, and a variety of unique biological organisms from diverse sources.
Developing efficient and practical bioprocesses frequently involves testing a large number of different strains and environmental conditions in various combinations. Although the ultimate goal is to identify an appropriate strain and conditions for production on an industrial scale (e.g., in a bioreactor with a 1,000-300,000 liter volume), bioprocess development begins on a much smaller scale. For example, screening of different strains is often conducted in microtiter plates, under relatively uncontrolled conditions and with only limited possibility of monitoring conditions during culture. After identification of strains that appear promising, further screening is performed in shaking flasks with a much larger volume (e.g., 25-100 ml). Such flasks typically allow only partial control over important environmental variables and cannot achieve the high oxygen (O2) concentrations typically used in large-scale fermentation processes. Thus the usefulness of these open loop systems for selecting the organism that will be optimal under actual bioprocess conditions is limited.
Scale-up to bench-scale, closed loop bioreactors, which offer improved control over environmental variables, increased oxygenation, and therefore the ability to achieve higher cell densities, is the next step. However, bench-scale reactors, with typical volumes of between 0.5 and 10 liters suffer from a number of drawbacks. Because of their large size, relatively high cost, and the time and effort required to obtain the data it is typically not practical to test as many combinations of strains and environmental conditions as would be desirable.
The inventors have recognized that there is a large technology gap between microtiter plates/flasks and closed loop controlled bioreactors. The gap is significant because its presence may allow potentially productive strains to be eliminated at the microtiter plate or shake-flask screening stage, due to optimization with respect to uncontrolled physical parameters, or it may allow potentially non-productive strains that do not perform well under typical industrial scale bioprocess conditions, e.g., high cell densities, to proceed to the next stage. There is thus a need in the art for a system to fill this gap. In particular, there is a need for small scale bioreactor systems that allow multiple experiments to be performed in parallel without an accompanying increase in cost, and that offer improved oxygen transfer capacity and control over environmental parameters, such as pH and dissolved oxygen.
Recent efforts to address the need for a parallel bioreactor system with the capabilities of a stirred tank reactor have focused on improving the oxygen transfer rate of microtiter plates (W. A. Duetz, L. Ruedi, R. Hermann, K. O'Connor, J. Buchs, and B. Witholt, Appl. Environ. Microb, 2000, 66, 2641-2646), improving the control capabilities of shake flasks (D. Weuster-Botz, J. Altenbach-Rehm, M. Arnold, Biochem. Eng. J, 2001, 7, 163-170), improving the parallelism of stirred tank bioreactors (J. Altenbach-Rehm, C. Nell, M. Arnold, and D. Weuster-Botz, Chem. Eng. Technol, 1999, 22, 1051-1058; S. R. Lamping, H. Zhang, B. Allen, P. Ayazi Shamlou, Chem. Eng. Sci., 2003, 58, 747-758; Y. Kostov, P. Harms, L. Randers-Eichhorn and G. P. Rao, Biotechnol Bioeng, 2001, 72, 346-352; P. Harms, Y. Kostov, J. A. French, M. Soliman, M. Anjanappa, A. Ram, and G. Rao, Biotechnol. Bioeng., 2006, 93, 6-13; R. Puskeiler, A. Kusterer, G. T. John and D. Weuster-Botz, Biotechnol. Appl. Biochem., 2005, 42, 227-235; R. Puskeiler, K. Kaufmann, D. Weuster-Botz, Biotechnol. Bioeng., 2005, 89, 512-523; D. Weuster-Botz, R. Puskeiler, A. Kusterer, K. Kaufmann, G. T. John, and M. Arnold, Bioprocess Biosyst Eng, 2005, 28, 109-119), or developing microfabricated bioreactor systems (M. M. Maharbiz, W. J. Holtz, R. T. Howe, and J. D. Keasling, Biotechnol. Bioeng., 2004,85, 376-381; F. K. Balagadde, L You, C. L. Hansen, F. H. Arnold, and S. R. Quake, Science. 2005, 309, 137-40; A. Zanzotto, N. Szita, P. Boccazzi, P. Lessard, A. J. Sinskey, and K. F. Jensen, Biotechnol. Bioeng., 2004, 87, 243-254; P. Boccazzi, A. Zanzotto, N. Szita, S. Bhattacharya, K. F. Jensen, A. J. Sinskey, Appl. Microbiol. Biotechnol, 2005, 68, 518-532; Z. Zhang, N. Szita, P. Boccazzi, A. J. Sinskey and K. F. Jensen, Proceedings of Micro Total Analysis Systems, Seventh International Conference on Miniaturized Chemical and Biochemical Analysis Systems, 765-768, Squaw Valley, Calif., USA, 2003; N. Szita, P. Boccazzi, Z. Zhang, P. Boyle, A. J. Sinskey and K. F. Jensen, Lab Chip, 2005, 5, 819-826). Each of these approaches has addressed parallelism, oxygenation, control, automation, and scalability to various degrees. Of these approaches, the miniature arrays of stirred tanks with robotic fluid handling have achieved the highest level of performance in terms of cell density and controlled parameters. However, these systems require expensive pipetting robotics and careful sterilization of the pipette tips to prevent contamination during frequent sampling.
The inventors have recognized that a microfabricated integration approach offers the potential for circumventing the need for robotic multiplexing, however, none of the microbioreactor systems developed to date have utilized microfluidic integration to achieve parallelism. In addition, no existing microfabricated approach has succeeded, even in a single reactor, in providing the oxygen transfer rate and the pH control capabilities of stirred tank bioreactors that are required for high cell density growth.
Current methods for pH control in miniature bioreactor arrays include diffusion of carbon dioxide gas into the liquid medium to generate carbonic acid, see, e.g., “Methods for intense aeration, growth storage, and replication of bacterial strains in microtiter plates,” W. A. Duetz, L. Ruedi, R. Hermann, K. O'Connor, J. Buchs, and B. Witholt, Appl. Environ. Microb. Vol. 66, pp. 2641-2646 (June 2000) and “A microfluidic pH-regulation system based on printed circuit board technology,” C. Laritz, and L. Pagel, Sensors and Actuators A, 84, 230 (2000), and generation of hydroxide or hydrogen ions by electrolysis of water, see, e.g., “A flow-through cell with integrated coulometric pH actuator,” S. Bohm, W. Olthuis, and P. Bergveld, Sensors and Actuators B, 47, 48 (1998) and “Micro-instruments for life science research,” B. van der Schoot, M. Boillat, and N. de Rooij, IEEE Transactions on Instrumentation and Measurement, 50, 1538 (2001). Carbon dioxide diffusion is limited because it cannot compensate for acids generated during microbial growth. While electrolytic methods can compensate for both acids and bases, unintended side reactions can generate unwanted compounds, and their implementation is complicated by the need to integrate a counter electrode with each growth chamber.
Furthermore, because diffusion of acids and bases to enable pH control is often slow at microbioreactor dimensions, controlling pH by simply injecting acid or base is not viable without something to facilitate mixing. Therefore, in order to realize a small scale bioreactor device that allows multiple experiments to be performed in parallel that offers improved oxygen transfer capacity and control over environmental parameters (such as pH and dissolved oxygen), a number of challenges need to be overcome.